Protein purification using displacement chromatography

Abstract

Disclosed herein are compositions and methods for the isolation and purification of proteins from a sample. In particular, the present invention relates to compositions and methods for isolating and purifying proteins incorporating a displacement chromatographic step. The present invention is also directed toward pharmaceutical compositions comprising one or more antibodies purified by a method described herein.

What is claimed is: 
     
       1. A method for producing a low acidic species human anti-TNFα antibody composition comprising a human anti-TNFα antibody, or antigen-binding portion thereof, wherein the human anti-TNFα antibody, or antigen-binding portion thereof, comprises a light chain variable region (LCVR) having a CDR1 domain, a CDR2 domain, and a CDR3 domain of adalimumab; and a heavy chain variable region (HCVR) having a CDR1 domain, a CDR2 domain, and a CDR3 domain of adalimumab, the method comprising:
 (a) contacting a first composition comprising the human anti-TNFα antibody, or antigen-binding portion thereof, with a chromatography media, wherein the first composition comprises more than 10% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof; 
 wherein the acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof, comprise species selected from the group consisting of charge variants, structure variants, fragmentation variants, and any combinations thereof, 
 wherein the acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof, do not include process-related impurities selected from the group consisting of host cell proteins, host cell DNA, and media components, and 
 wherein the human anti-TNFα antibody, or antigen-binding portion thereof, binds to the chromatography media; 
 (b) displacing the human anti-TNFα antibody, or antigen-binding portion thereof, bound to the chromatography media with a displacing buffer; and 
 (c) collecting a second composition comprising the displaced human anti-TNFα antibody, or antigen-binding portion thereof, wherein the second composition comprises less than 10% total acidic species of human anti-TNFα antibody, or antigen-binding portion thereof, 
 thereby producing a low acidic species human anti-TNFα antibody composition. 
 
     
     
       2. The method of  claim 1 , wherein the chromatography media is an ion exchange adsorbent material. 
     
     
       3. The method of  claim 2 , wherein the ion exchange adsorbent material is an anion exchange adsorbent material. 
     
     
       4. The method of  claim 2 , wherein the ion exchange adsorbent material is an cation exchange adsorbent material. 
     
     
       5. The method of  claim 4 , wherein the cation exchange (CEX) adsorbent material is selected from the group consisting of a CEX resin and a CEX membrane adsorber. 
     
     
       6. The method of  claim 1 , wherein the chromatography media is a multimodal adsorbent material comprising cation exchange and hydrophobic interaction functional groups. 
     
     
       7. The method of  claim 1 , wherein the pH of the displacing buffer is lower than the isoelectric point of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       8. The method of  claim 7 , wherein the pH of the displacing buffer is about 6.0 to about 8.0. 
     
     
       9. The method of  claim 1 , wherein the displacing buffer carries positive charge. 
     
     
       10. The method of  claim 1 , wherein the conductivity of the displacing buffer is about 2 to about 20 mS/cm. 
     
     
       11. The method of  claim 1 , wherein the chromatography media is present in a column having a length of about 10 cm to about 30 cm and wherein flow residence time is about 5 min to about 25 min. 
     
     
       12. The method of  claim 1 , wherein displacing the human anti-TNFα antibody, or antigen-binding portion thereof, bound to the chromatography media comprises using a first displacing buffer followed by using a second displacing buffer. 
     
     
       13. The method of  claim 1 , wherein displacing is achieved using a multiple-step displacement, a two-step displacement, or a linear displacement. 
     
     
       14. The method of  claim 1 , wherein the displacing buffer comprises protamine sulfate. 
     
     
       15. The method of  claim 14 , wherein the displacing buffer comprises 0.1 to 5 mM protamine sulfate. 
     
     
       16. The method of  claim 15 , wherein the displacing buffer comprises 0.25 mM protamine sulfate. 
     
     
       17. The method of  claim 1 , wherein the displacing buffer comprises a quaternary ammonium salt. 
     
     
       18. The method of  claim 17 , wherein the displacing comprises 0.1 to 10 mM quaternary ammonium salt. 
     
     
       19. The method of  claim 18 , wherein the displacing buffer comprises 0.5 mM quaternary ammonium salt. 
     
     
       20. The method of  claim 1 , wherein one displacing buffer is used. 
     
     
       21. The method of  claim 1 , wherein the chromatography media is a mixed mode media. 
     
     
       22. The method of  claim 1 , wherein the second composition comprises less than 9% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       23. The method of  claim 1 , wherein the second composition comprises less than 8% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       24. The method of  claim 1 , wherein the second composition comprises less than 7% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       25. The method of  claim 1 , wherein the second composition comprises less than 6% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       26. The method of  claim 1 , wherein the second composition comprises less than 5% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       27. The method of  claim 1 , wherein the second composition comprises less than 4.5% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       28. The method of  claim 1 , wherein the second composition comprises less than 4% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       29. The method of  claim 1 , wherein the second composition comprises less than 3% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       30. The method of  claim 1 , wherein the second composition comprises less than 1.4% total acidic species of the human anti-TNFα antibody, or antigen-binding portion thereof. 
     
     
       31. The method of  claim 1 , wherein the human anti-TNFα antibody, or antigen-binding portion thereof, is adalimumab, or an antigen-binding portion thereof.